Project Summary/Abstract At present there are more than 30,000 patients waiting to receive liver transplants. The number is increasing due to an aging US population accompanied by an increasing incidence of chronic liver diseases associated with such disorders as alcoholic liver disease, hepatitis, NAFLD and NASH. In spite of efforts to persuade people to serve as organ donors, the demand increasingly outstrips the supply for organ transplantation. One solution to this problem is the ability to generate human livers in animals for liver as well as hepatocyte transplantation. Although there are numerous protocols to differentiate human embryonic stem cells (hESCs), and inducible pluripotent stem cells (iPSCs) ex vivo to a variety of cell types, they have encountered significant challenges in translation to the clinic. However, it is now possible to regenerate the replica of organs/cells from one species of animal within the body of a second species. This involves the knockout (KO) of specific developmental genes in the blastocyst of species two; and the intra-blastocyst injection of pluripotent stem cells from species one to generate offspring that carry organs/cell types derived from that donor. The translation of this approach requires an efficient gene-editing technology. In fact, novel TALEN/CRISPR/Cas9 technologies provide such a rapid, and cost-effective means to generate genetically modified animals. Accordingly, we propose to employ the TALEN/CRISPR technology to knockout specific genes associated with liver development in the pig blastocyst. We hypothesize that non-human primate liver can be generated in the pig by the injection of marmoset embryonic stem cells (ESCs) into TALEN and/or CRISPR-KO porcine blastocysts. Marmosets are often used for research on human aging and disease because their bodies are very close to those of humans. We have designed three Specific Aims to test our central hypothesis. Specifically, we will (1) generate pig-pig liver chimeras by blastocyst complementation as proof-of-principle; (2) develop marmoset livers in a model of non-human primate-pig chimeras in addition to characterizing their functionality; and (3) interrogate the genetic programs involved in generating human-porcine chimeric livers. The generation of whole livers that are comprised primarily of non-human primate hepatic cells derived from implanted marmoset ESCs would represent a paradigm shift and provide the necessary preclinical evidence for ultimately creating human livers in animals. If successful, the proposed research would be a game-changer that could conceivably pave the way for the production of human livers in large animals, such as the pig, for organ and/or hepatocyte transplantation that is specifically tailored for each patient suffering from a chronic liver disease. In addition, this novel, albeit somewhat high-risk approach circumvents many of the problems associated with decades of research on xenotransplantation. The potential impact on improved health care in the U.S. and world wide for liver diseases is great.